Hua Su Professor Center for Cerebrovascular Research, Departments of Anesthesia and Perioperative Care, University of California, San Francisco, California, USA. Impact of Bone Fracture on Stroke Recovery

1.Stroke is the leading cause of disability in adults and an important risk factor for bone fracture. In the United States, approximately 70,000 stroke victims suffer from bone fracture within the first year after their stroke. 2. Alternatively, stroke is one of the most devastating complications of bone fracture. About 0.2% to 4.1% patients suffer from stroke after hip surgery. 3. The impact of bone fracture on stroke recovery has not been fully studies. Background Sennerby, JAMA, 2009 Kanis, Stroke, 2001 Lakshminarayan, Stroke, 2011

Tibia fracture increase systemic and hippocampus inflammation and resulted in cognitive decline Terrando et al. PNAS, 2010

pMCAO D0 Bone fracture D1 D3 Behavioral test Sample collection Behavior training (4 days) Bone fracture after stroke

Adhesive removal test Bouet, et al, Nature protocol,2009 Adhesive tape (0.3 X 0.3 cm) was applied on each paw of the mouse. The time was recorded with a maximum testing time of 120 seconds when the tapes were removed from each paw.

Corner test Zhang, et al, Journal of Neuroscience, 2002 When mice come near the corner, both sides of their vibrissae are stimulated. The mice will rear forward and upward, then turn back to face the open end. Normal mice would turn to the left or right side with equal frequency, whereas the stroke mice would turn more frequent to the lesion side (left).

Tibia fracture one day after pMCAO increased neuroinflammation and behavioral dysfunction Degos, et al, Anesthesiology, 2012

Tibia fracture one day after pMCAO increased neuronal injury Degos, et al, Anesthesiology, 2012

Depletion of macrophage reduces the negative impact of bone fracture on stroke injury

Bone fracture before stroke

Tibia fracture 6 hours or one day before pMCAO increased neuronal injury

Tibia fracture 6 hours or one day before pMCAO increased neuroinflammation

Tibia fracture 6 hours or one day before pMCAO increased behavioral dysfunction

Bone fracture Border zone Healing stroke lesion Macrophages  Core Toxic processes chemokines Unknown alarmins Macrophages activation activation HMGB1 Bone fracture exacerbates stroke injury through enhance inflammation Degos, et al, Anesthesiology, 2012

Cholinergic inflammation resolving Pathway Su et al. Molecular Medicine, 2012

Design Groupdosenotelabel Saline PHA 0.4 mg/kgInjection 1 timePHA-L 0.8 mg/kgInjection 1 timePHA-H1 0.8 mg/kgInjection 2 timesPHA-H2 MLA 4 mg/kgInjection 1 timeMLA-L 6 mg/kgInjection 1 timeMLA-H1 6 mg/kgInjection 2 timesMLA-H2 PHA (PHA568487): α 7 agonist MLA (methyllycaconitine): α 7 antagonist

Experimental design Han et al. Journal of Neurochemistry, 2014

Activation of  -7 nAchR reduces behavioral deficits

Han et al. Journal of Neurochemistry, 2014 Activation of  -7 nAchR reduces reduced infarct volume and apoptotic neurons

Han et al. Journal of Neurochemistry, 2014 Activation of  -7 nAchR decreased microglia/macrophages in the peri-infarct region

Han et al. Journal of Neurochemistry, 2014 Activation of  -7 nAchR decreased reduced M1 and increased M2 marker gene expression

Han et al. Journal of Neurochemistry, 2014 Activation of  -7 nAchR increased anti-oxidant gene expression and reduced NADPH oxidase

Han et al. Journal of Neurochemistry, 2014 Activation of  -7 nAchR reduced nuclear factor kappa b (NF-kb) activity

oxidative stress microglia/ macrophages α-7 nAchR agonist (PHA ) anti-oxidative gene oxidative gene + Increase brain injury M1/M2 M1 M2 M1/M2 Reduce brain injury Summary Han et al. Journal of Neurochemistry, 2014

1. Bone fracture shortly before or after stroke increase stroke related neuronal injury through enhance inflammation. 2. Activation of  7 nAChR improves ischemic stroke recovery and reduces the adverse effect of bone fracture on stroke recovery through inhibition of inflammation and oxidative stress Summary

Thank You Lei Zhan Wan Zhu Rui Zhang Dingquan Zou Chen Bao Ethan Winkler Alireza Sadighi Zhao Li Funding: NIH AHA Michael Ryan Zodda Foundation Espen Walker Wanqiu Chan Eunjung Choi Fanxia Shen Yi Guo Lei Mao Marine Camus Mamta Wankhede Vencent Dego Zhengyi Han Yue He Cameron McDougall Liang Wang Shuai Kang

Vagus nerve stimulation reduces infarct size in rat and mouse focal cerebral ischemia Cholinergic anti-inflammatory pathway inhibits cytokine release through a mechanism that requires the  7 subunit-containing nAChRs. Anti-inflammation Borovikova et al. Nature, 2000 Bernik et al. J. Vasc. Surg Wang et al. Nature, 2003 Ay et al. Brain Research, 2011 Han et al. PLOS One, 2014